Bottom Line:
In addition to deregulated T cell growth and survival, transgene expression impairs the development of normal T cell populations as evidenced by diminished numbers of TCRhi CD8 single-positive thymocytes.This defect was significantly amplified in the periphery and was accompanied by a decrease in CD4(+) T cells.Taken together, these in vivo findings indicate that the NF-kappaB/Rel signaling pathway contains compensatory components that are essential for the establishment of normal T cell subsets.

ABSTRACTMembers of the nuclear factor (NF)-kappaB/Rel family transcription factors are induced during thymic selection and in mature T lymphocytes after ligation of the T cell antigen receptor (TCR). Despite these findings, disruption of individual NF-kappaB/Rel genes has revealed no intrinsic defect in the development of mature T cells, perhaps reflecting functional redundancy. To circumvent this possibility, the T cell lineage was targeted to express a trans-dominant form of IkappaBalpha that constitutively represses the activity of multiple NF-kappaB/Rel proteins. Transgenic cells expressing this inhibitor exhibit a significant proliferative defect, which is not reversed by the addition of exogenous interleukin-2. Moreover, mitogenic stimulation of splenocytes leads to increased apoptosis of transgenic T cells as compared with controls. In addition to deregulated T cell growth and survival, transgene expression impairs the development of normal T cell populations as evidenced by diminished numbers of TCRhi CD8 single-positive thymocytes. This defect was significantly amplified in the periphery and was accompanied by a decrease in CD4(+) T cells. Taken together, these in vivo findings indicate that the NF-kappaB/Rel signaling pathway contains compensatory components that are essential for the establishment of normal T cell subsets.

Figure 3: Failure of IL-2 to rescue the proliferative defect in IκBα(ΔN) mice. Thymocytes from either NTg (open bars) or Tghi (closed bars) mice were treated for 40 h with (A) combinations of PMA (50 ng/ml) and ionomycin (1 μg/ml). (B) Con A (2.5 μg/ml), or (C) plate-bound anti-CD3 (10 μg/ml), in the presence or absence of IL-2 (100 U/ml) as indicated. Cells were pulsed for an additional 8 h with tritiated thymidine and harvested for scintillation counting. The results are shown as the mean of tritiated thymidine incorporation (± SEM) for eight Tghi mice and eight NTg littermates (four independent experiments). (D) Splenocytes depleted of B cells and macrophages were analyzed under similar conditions using plate-bound anti-CD3 (10 μg/ml), agonistic antibodies against CD28 (10 μg/ml), and IL-2 (100 U/ml) as indicated.

Mentions:
Prior studies have shown that c-Rel–deficient T cells develop normally but exhibit impaired mitogenic responses (14). This proliferative defect was fully reversed by exogenous IL-2 (14). Evidence of a transgene-dependent block in IL-2 production by Tghi thymocytes led us to investigate their proliferative response to the T cell mitogens Con A, combinations of PMA and ionomycin, or immobilized antibody to the TCR (anti-CD3). As shown in Fig. 3, Tghi thymocytes manifested a dramatic decrease in the proliferation induced by each of these mitogens (A–C; control). We reasoned that this growth defect might be attributed to the 20-fold difference in IL-2 concentrations in these cultures (see Fig. 2). However, as shown in Fig. 3 A addition of exogenous IL-2 to Tghi thymocytes failed to restore proliferation to normal levels. Similar results were obtained with unfractionated (data not shown) or B cell–depleted splenocytes (Fig. 3 D). These results are in sharp contrast with those obtained using c-Rel–deficient mice (14), presumably reflecting an additional contribution by RelA (Fig. 1 D). In this regard, there was a 50% decrease in the frequency of T cells positive for IL-2Rα chain (CD25), which has in its 5′-flanking DNA a site for RelA/p50 binding (5, 33). However, Tghi-derived cells that were able to undergo blast transformation and enter S phase expressed normal levels of CD25 (data not shown). We conclude that the failure of exogenous IL-2 to restore normal proliferation of Tghi T cells is likely due to the reduced expression of a competence factor other than the IL-2Rα chain.

Figure 3: Failure of IL-2 to rescue the proliferative defect in IκBα(ΔN) mice. Thymocytes from either NTg (open bars) or Tghi (closed bars) mice were treated for 40 h with (A) combinations of PMA (50 ng/ml) and ionomycin (1 μg/ml). (B) Con A (2.5 μg/ml), or (C) plate-bound anti-CD3 (10 μg/ml), in the presence or absence of IL-2 (100 U/ml) as indicated. Cells were pulsed for an additional 8 h with tritiated thymidine and harvested for scintillation counting. The results are shown as the mean of tritiated thymidine incorporation (± SEM) for eight Tghi mice and eight NTg littermates (four independent experiments). (D) Splenocytes depleted of B cells and macrophages were analyzed under similar conditions using plate-bound anti-CD3 (10 μg/ml), agonistic antibodies against CD28 (10 μg/ml), and IL-2 (100 U/ml) as indicated.

Mentions:
Prior studies have shown that c-Rel–deficient T cells develop normally but exhibit impaired mitogenic responses (14). This proliferative defect was fully reversed by exogenous IL-2 (14). Evidence of a transgene-dependent block in IL-2 production by Tghi thymocytes led us to investigate their proliferative response to the T cell mitogens Con A, combinations of PMA and ionomycin, or immobilized antibody to the TCR (anti-CD3). As shown in Fig. 3, Tghi thymocytes manifested a dramatic decrease in the proliferation induced by each of these mitogens (A–C; control). We reasoned that this growth defect might be attributed to the 20-fold difference in IL-2 concentrations in these cultures (see Fig. 2). However, as shown in Fig. 3 A addition of exogenous IL-2 to Tghi thymocytes failed to restore proliferation to normal levels. Similar results were obtained with unfractionated (data not shown) or B cell–depleted splenocytes (Fig. 3 D). These results are in sharp contrast with those obtained using c-Rel–deficient mice (14), presumably reflecting an additional contribution by RelA (Fig. 1 D). In this regard, there was a 50% decrease in the frequency of T cells positive for IL-2Rα chain (CD25), which has in its 5′-flanking DNA a site for RelA/p50 binding (5, 33). However, Tghi-derived cells that were able to undergo blast transformation and enter S phase expressed normal levels of CD25 (data not shown). We conclude that the failure of exogenous IL-2 to restore normal proliferation of Tghi T cells is likely due to the reduced expression of a competence factor other than the IL-2Rα chain.

Bottom Line:
In addition to deregulated T cell growth and survival, transgene expression impairs the development of normal T cell populations as evidenced by diminished numbers of TCRhi CD8 single-positive thymocytes.This defect was significantly amplified in the periphery and was accompanied by a decrease in CD4(+) T cells.Taken together, these in vivo findings indicate that the NF-kappaB/Rel signaling pathway contains compensatory components that are essential for the establishment of normal T cell subsets.

ABSTRACTMembers of the nuclear factor (NF)-kappaB/Rel family transcription factors are induced during thymic selection and in mature T lymphocytes after ligation of the T cell antigen receptor (TCR). Despite these findings, disruption of individual NF-kappaB/Rel genes has revealed no intrinsic defect in the development of mature T cells, perhaps reflecting functional redundancy. To circumvent this possibility, the T cell lineage was targeted to express a trans-dominant form of IkappaBalpha that constitutively represses the activity of multiple NF-kappaB/Rel proteins. Transgenic cells expressing this inhibitor exhibit a significant proliferative defect, which is not reversed by the addition of exogenous interleukin-2. Moreover, mitogenic stimulation of splenocytes leads to increased apoptosis of transgenic T cells as compared with controls. In addition to deregulated T cell growth and survival, transgene expression impairs the development of normal T cell populations as evidenced by diminished numbers of TCRhi CD8 single-positive thymocytes. This defect was significantly amplified in the periphery and was accompanied by a decrease in CD4(+) T cells. Taken together, these in vivo findings indicate that the NF-kappaB/Rel signaling pathway contains compensatory components that are essential for the establishment of normal T cell subsets.